ETA (Otis) Here's a link to a MS Word document that is reasonably formatted which makes cutting and pasting easier, etc. http://www.sendspace.com/file/0wsvpr. Scroll to the bottom of the page and click the download button. You may get a popup - just close it if you do. That's the price for a "free" service.

Anybody able to de-encrypt this puppy. I would love to copy and paste the DERSE cells abstracts since it deals with measuring titers in XMRV positive patients (no matter what Dr. Coffin says, bleh) but I can't break the encryption and it won't let me copy and paste. (sad dog eyes)

MMMMhmmm, check out the Detection of XMRV sequences in EBV-transformed B cell lines. (page 24 in the presentation section)

The study was done in Barcelona, Spain the presentation is listed under Pathogenensis!

Background: XMRV infection has been found in humans linked with prostate cancer and chronic fatigue syndrome (CFS). XMRV is able to infect a wide range of cells and has been found in a variety of cells types both in Vitro and in vivo, including B cells and other immune cell types.

Click to expand...

I won't go into the methodology except to say it was a small sample group and I would like to see this in a much larger group and read the methods in depth.

Conclusions: Despite the discrepancies observed in the different PCR approaches using gag, pol or env sequences, our data suggest that EBV transformed B cell lines harbor XMRV specific sequences, and therefore this cell type may represent a reservoir for XMRV contributing to its potential pathogenesis.

I think the sausage is pretty well ground, lets say we make patties now

Here's another good one

Looks like there may be a race to look at the inflammatory response. The Dr.'s White and Dr. Klimas have been plugging away in this field for a while now. Personally I hope they publish pretty quick and get the glory! (big grins) Here is another study confirming what those guys all read know.

Background: In order to study the host response to XMRV infection at the RNA level, we used PCR array to analyze the expression of a focused panel of genes related to the inflammatory response involved in XMRV infection. The PCR array is a set of optimized real time PCRA primer assays on 96-well plates for pathway or disease focused genes together with appropriate RNA quality controls that are capable of performing gene expression analysis.

Results: Cytokines, chemokines and chemokine receptors involved in host inflammatory response like IL-1 (alpha and beta), IL-13, IL-17C TNF-alpha, CCL5, CCL17 and CCL19 were found to be upregulated at the time points tested. The gene expression profiles of IL-8, CCL2, CCL29, CXCL3, CXCL5 and CCR7 were found to be significantly down-regulated. Other genes involved in signal transduction pathways like FOS, IGFBP3, EGR1 and WNT were differentially regulated at the time points tested.

There's another confirmation study here that I hadn't heard about - Hanson from Cornell with Dr David (I assume? Dept of Pediatrics at Buffalo?) Bell looking at 10 severe CFS, 10 "recovered" CFS and 10 healthies. XMRV in 80%, 30% and 10% of plasma samples, respectively. They report that this confirms the Lombardi findings.

Have I had my head in a bag or something? I haven't seen any mention of this anywhere!

cant wait for the Uk study to be published and finding out what the uk positive % actually is, says 60% here, Mindy said 70% and even higher has been mentioned....exciting times.

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It says ">60%" which I thought odd; it's normal to give the exact percentage. Does anyone have any idea why it's reported in this way? Apologies if I'm missing something obvious, my eyes are bouncing around the text at the moment due to an inability to concentrate properly (back to Midsomer Murders again, then).

Human immunodeficiency virus (HIV) can be estimated using indicator cell lines, such as GHOST cells, within days of infection. HIV indicator cells rely on production of Tat to transactivate expression of a reporter gene under the control of HIV LTR sequences. Simple retroviruses typically do not encode transcriptional transactivators. For simple retroviruses that lack transformational or cytopathic activity, their titers are often measured by infection of cells after end point dilution and assaying for virus proliferation after weeks of culture. Replication-independent vectors have been leveraged to assay the mobilization of retrotransposable elements and the replication of retroviruses. Here we describe an indicator cell line for the detection of infectious murine leukemia virus-related virus (XMRV) that relies on the propagation of a vector, which leads to the expression of a GFP reporter.

Materials and methods

We constructed an MLV vector encoding puromycin resistance and a CMV enhancer/promoter driven GFP reporter gene whose transcription was antisense to the vector mRNA. The GFP reporter sequence (iGFP) was interrupted by an intron placed in the sense direction relative to the vector. The prostate cell line, LNCaP, was stably transfected with the above construct, and puromycin-resistant cell clones were obtained and assayed for sensitivity to XMRV via transduction and activation of GFP. GFP was detected by flourescence microscopy or FACS analysis of paraformaldehyde-fixed indicator cells. XMRV was obtained from supernatants of CWR22Rv1 cells or HEK 293 T cells transfected with the pVP62 molecular clone.

Results

Several LNCaP-iGFP cell clones displaying sensitivity to XMRV infection after end point dilution were isolated and designated Detectors of Exogenous Retroviral Sequence Elements (DERSE) cells. GFP signal could be detected within three days of infection, with the number of GFP-positive cells increasing over subsequent days. Infection was enhanced by the addition of polybrene to cultures. Plasma and serum samples from persons previously found positive for XMRV detectably infected the DERSE cells; whereas samples from XMRV-negative individuals did not induce GFP expression in the cells even after weeks of culture. Compared to detection of infection using LNCaP parental cells, XMRV from patient samples was observed two to three times faster in DERSE cells. GFP signal after virus inoculation was dose-dependent and could be impaired by heat inactivation of virus stocks or the addition of AZT to cultures at the time of infection.

Conclusions
Here we describe DERSE cells, a new indicator cell system for the sensitive and rapid detection of replicating XMRV. In principle, DERSE cells should also detect other gammaretroviruses capable of infecting human cell lines. Because this indicator cell system utilizes GFP as a reporter, infection can be monitored in live cultures by florescence microscopy. Consistent with prior reports, using LNCaP cells, DERSE cells detect virus from human blood samples.

This should provide a titer number that can be used in testing ARV trials at least enough to say that there is improvement or not improvement. So NANA

Background: XMRV infection has been found in humans linked with prostate cancer and chronic fatigue syndrome (CFS). XMRV is able to infect a wide range of cells and has been found in a variety of cell types both in Vitro and in vivo, including B cells and other immune cell types.

Materials and methods: In order to determine the presence of XMRV sequences in B cells, we have screened 21 B cell lines available in our laboratory. These cells were generated by EBV immortalization of PBMC obtained from 11 CFS affected individuals (fulfilling both Fukuda and Canadian criteria), 5 healthy donors, 4 HIV infected individuals and 1 prostate cancer patient- DNA was extracted from dry cell pellets and XMRV sequences were amplified using a real-time PCR covering a 150bp pol sequence and using a nested approach in both gag and Env genes.

Results: Envelope amplification yielded positive bands in 4 out of 21 individuals tested, 3 CFS affected individuals and 1 healthy donor. However, gag amplihcation yielded only 3 positive samples (1 CFS affected individual, 1 healthy donor and one HlV+ patient). In contrast, Real-time PCR of Pol fragment detected 7 positives samples in 14 individuals tested (4 SFC , 2 donors and 1 HN+ individuals). To confirm the presence of XMRV sequences we performed sequence analyses of gag and env amplicons. The analysis of the three available gag sequences confirmed the XMRV characteristic 24-nt deletion, which is not found in any known exogenous MuLV. Sequences were 100% identical to reported XMRV sequences. Furthermore, envelope sequences
were also homologous to previously described XMRV sequences. ln this case, sequence variability was low or absent Interestingly, most of changes observed corresponded to G to A mutations that were accumulated in one positive sample.

Conclusions: Despite the discrepancies observed in the different PCR approaches using gag, pol or env sequences, our data suggest that EBV transformed B cell lines harbor XMRV
specific sequences, and therefore this cell type may represent a reservoir for XMRV contributing to its potential pathogenesis.

NB. If someone uses the OCR tool, you'd get a better result (less spelling errors) if you have a large/high def screen.

Cornell University, Dept of Molecular Biology and Genetics, Ithaca NY, USA; State University of New York, Dept of Medical Anthropology, Buffalo NY, USA; Cornell University, School of Operations Research and Information Engineering, Ithaca NY, USA; State University of New York, dept of Pediatrics, Buffalo NY, USA

Background:

In October 2009, Lombardi et all repoted detection of XMRV in blood of persons with CFS. Since then, four studies that performed PCR analysis on DNA from blood of CFS patients failed to detect XMRV. The present blinded study was undertaken to determine whether XMRV could be detected in peripheral blood mononuclear cells (PBMCs) from three small groups of subjects from a single geographic area.

Materials and methods:

The 30 adult subjects of this study were divided into three groups of ten persons each: severely ill with CFS, recovered from CFS, and a control group lacking a CFS diagnosis at any time. Inclusion did not require that they became ill in the time frame or in the exact location of a cluster of CFS in Lyndonville, NY. All patients in group 1 ("severe CFS") currently meet Fukuda criteria. The individuals in group 2 ("recovered CFS") had met Fukuda criteria for CFS at one time in the past, but presently considered themselves recovered. One patient in group 1 and seven in group 2 are considered part of the "Lyndonville outbreak". The ten persons included in group 3 "healthy, non-household contact controls" have never experienced a CFS-like illness, are healthy, and have never lived with persons with CFS. Average ages of the three groups are 42.2, 39, 49.8, respectively; and M:F ratio is 2:8 6:4, 2:8. All thirty subjects completed seven different clinical survey instruments including the SF-36. Blood was collected in EDTA tubes and PBMCs and plasma separated within 24 hours of draw. Plasma from some samples was incubated with LNCaP cells. The Invitrogen Superscript (R) VILO (TM) cDNA Synthesis Kit was used to produce cDNA from RNA isolated from PBMCs or LNCaP cells. Nested PCR with USB Hot-Start IT FideliTaq was performed using the Gag O-Gag l primers originally described by Urisman et al. PCR products were separated on gels and any bands of approximately 400 bp were sequenced and analyzed by BLAST for similarity to the XMRV gag gene. PCR with mouse COX2 primers was performed on all cDNA preparations to rule out mouse cell contamination.

Results:

XMRV gag sequences were detected in eight of the "severe CFS", three of the "recovered CFS" and in one of the controls. Plasma from six blood samples, five CFS and one control, was incubated with LNCaP cells; the six cultures were passaged six times. The five cultures found to exhibit XMRV gag sequences were those inoculated with CFS patient plastma. Although group 2 members described themselves as recovered, their scores on the SF-36 were significantly lower than the healthy control group, according to Hoteling's T2 test. Tukey's multiple comparison of means indicates that there are highly significant differences between the scores of the "severe" and controls on all 7 instruments.

Thanks, Mojoey - that's the one I mentioned earlier. I thought it was really exciting - another confirmation study - so why is it so quiet in here???! Is this thread invisible or something? Or have we just got blase now...

Actually, would anyone care to comment on the higher prevalence of XMRV in the plasma of present vs "recovered" CFSers in the study (8/10 vs 3/10)? It's clear that the "recovered" weren't fully recovered, according to the description of their physical functioning.

Oddly, 1/10 of the present CFS group and 7/10 of the "recovered" were from the Lyndonville outbreak - somehow I'd have expected Lyndonville people to be more likely to be XMRV+.

Small numbers of course but if they generalised, would it be possible for all/most of the CFS people to be XMRV+ to a similar degree whether "recovered" or not but for XMRV to be less likely to be detected in plasma in the recovered group? And if so, why?